Randomly Ordered Arrays and Methods of Making and Using

ABSTRACT

Arrays including microparticles having probe and marker moieties are used for the detection of a target in a sample. Microparticles are randomly immobilized on at least a portion of a substrate. A detection scheme is performed to detect the marker associated with the microparticle and the identity of the probe, and any target bound to the probe.

REFERENCE TO CO-PENDING APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 09/972,687, entitled RANDOMLY ORDERED ARRAYS AND METHODS OFMAKING AND USING, filed Oct. 5, 2001, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to the field of arrays for use in detecting atarget suspected to be present in a sample. More particularly, theinvention relates to arrays utilizing microparticles containing aself-encoding marker.

BACKGROUND OF THE INVENTION

In the past several years, a new technology, called the DNA array, hasattracted interest among biologists. This technology promises to monitorpart or all of an organism's genome on a single chip so that researcherscan develop a better picture of the interactions among hundreds orthousands of genes simultaneously. This technology has been termedbiochip, DNA chip, DNA microarray, gene array, and genome chip.Generally, a DNA array relies upon standard base pairing rules developedby Watson and Crick to analyze the presence, or the sequence, of aparticular complementary nucleic acid sequence.

More recently, attention has focused on fabrication of protein orpeptide arrays, and this area is commonly referred to as “proteomics.”In one example of this approach, a library of peptides can be used asprobes to screen for drugs. The peptides can be exposed to a receptor,and those probes that bind to the receptor can be identified. In oneapplication, more than 10,000 protein spots were printed on a glassslide. The chip was used to identify protein-protein and protein-druginteractions (G. MacBeath and S. L. Schreiber, 2000, Printing Proteinsas Microarrays for High-Throughput Function Determination, Science189:1760-1763).

In more recent years, the demand for high-throughput and cost-effectiveanalysis of complex mixtures has driven technology toward thefabrication of compact, high-density array devices. These arrays arefabricated using conventional techniques such as ink-jet printing,screen printing, photolithography, and photodeposition, in which thesensing chemistries are applied directly to the sensor surface.Typically, an array is fabricated by attaching a nucleic acid or peptidedirectly to a substrate. Multiple fabrication steps are commonlyrequired that are labor intensive and subject to some degree ofvariability.

Given current fabrication schemes, the precise location of a probe onthe surface of an array must be known prior to interrogating a sample.Therefore, fabrication of the arrays relies upon such techniques asprinting or spotting of the probe onto the surface of the array, so thatthe addresses or locations of each probe is known prior to use of thearray. Once the complexes are detected, the location of the complex iscompared to the mapped surface of the array, and the identity of thetarget is determined.

SUMMARY OF THE INVENTION

The invention generally relates to detecting a target in a sample usingan array of probes. More specifically, a target can be detecting usingan array that includes a substrate and a plurality of microparticles,which are coupled to probes, randomly immobilized on the substrate. Eachmicroparticle includes a probe and a self-encoding marker which forms aunique self-encoding marker/probe pair on the microparticle. Theplurality of microparticles having unique self-encoding marker/probepairs are immobilized on the substrate via an immobilization material.Detection of the target in a sample can be accomplished by applying asample suspected of containing the target to the array, allowing thetarget to bind to the probes coupled to the microparticles, and thendetecting a target marker coupled to the target and detecting the selfencoding markers of microparticles having unique self-encodingmarker/probe pairs. Although the microparticles are randomly located onat least a portion of the array, the presence and identity of the targetcan be determined by the self-encoding marker/probe pairs.

In some embodiments the immobilization material includes a reactivepolymer; preferably the reactive polymer is a photoreactive polymer orcopolymer. In other embodiments the immbolization material includes abinding pair.

In some embodiments the array includes multiple arrays, subarrays, orcombinations thereof. An array having subarrays can be prepared bycreating particular subsets of microparticles having uniqueself-encoding marker/probe pairs. In some embodiments the detection ofthe target is accomplished by determining a particular subset ofmicroparticles according to its location on the array in combinationwith detection of the self-encoding marker/probe pairs.

The self encoding marker can include at least one detectable particlewhich can include fluorophores, quantum dots, radioisotopes, andmagnetic particles. Combinations of these detectable particles can beused to provide a unique self-encoding marker.

The invention also includes methods of fabricating the arrays and thearrays themselves.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an array and a method of preparing thearray.

FIG. 2 is a schematic diagram of an array and a method of preparing thearray.

FIG. 3 is a schematic diagram of a method for preparing subsets ofmicroparticles having self encoding marker/probe pairs and a method ofpreparing subarrays.

FIG. 4 is a schematic diagram of an array and a method of preparing thearray.

FIG. 5 is a photomicrograph of a portion of an array containingmicroparticles.

FIG. 6 is a photomicrograph of a portion of an array containingmicroparticles.

FIG. 7 is a schematic diagram of subarrays and a method for preparingthe subarrays.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein, “array” refers to a plurality of different probemolecules presented on a surface, where unique probe molecules arecoupled to individual microparticles and the microparticles are randomlyimmobilized on a substrate. Each microparticle is also associated with aself-encoding marker thereby forming unique self-encoding marker/probepairs, enabling detection of the individual microparticles and theidentity of the probe. An array can present one set of microparticleshaving unique self-encoding marker/probe pairs or can present more thanone set or more than one subset of microparticles having uniqueself-encoding marker/probe pairs thereby allowing multiple arrays,subarrays, or combinations thereof, on the substrate.

A “set” of microparticles having unique self-encoding marker/probe pairsgenerally refers to a plurality of microparticles having uniqueself-encoding marker/probe pairs wherein the plurality of microparticlespresents all of the probe molecules to be arrayed on the substrate.

As used herein, “multiple array” refers to an array, that presents morethan one set or subset of microparticles having unique self-encodingmarker/probe pairs disposed at defined locations on the substrate.

As used herein, “subarray” refers to a portion of an array that containsa subset of microparticles which having a plurality of microparticleshaving unique self-encoding marker/probe pairs wherein the plurality ofmicroparticles presents a portion of all the probe molecules arrayed onthe substrate. An array typically includes more than one subarray, eachsubarray being different.

As used herein, “random” or “random distribution” or “randomly arrayed”refers to the arbitrary localization of an immobilized microparticlehaving a unique self-encoded marker/probe pair on a substrate by themethod of the invention. For a particular set or subset ofmicroparticles having unique self-encoded marker/probe pairs thelocalization of an individual microparticle having a unique self-encodedmarker/probe pair within the set or subset is random on the portion ofthe substrate on which the set or subset is disposed. A particularmicroparticle having a having a unique self-encoded marker/probe paircan be randomly immobilized on the surface of a substrate even thoughthe microparticles may be directed to patterned portions of thesubstrate. Therefore, microparticles can be ordered according toposition on the substrate although the arrangement according to theunique self-encoded marker/probe pairs is random. Such embodiments arediscussed herein. The location of a microparticle having a uniqueself-encoded marker/probe pair within the immobilized set or subset isnot determined until a detection step is performed.

As used herein, a “probe” is a moiety that is immobilized on a substrateto form an array. Typically, according to the invention, the probe iscoupled to a microparticle, and the microparticle is immobilized on asubstrate, thereby forming a portion of an array. The probe is amolecule, a particle, or a cell that can specifically interact with aparticular target. The probe can include naturally occurring or man-mademolecules, and it can be used in its unaltered state or as aggregateswith other species. Typically the specific interaction of the probe andthe target is based on chemical bonds that establish affinityinteractions between the probe and target, for example, between anantibody and an antigen, or specific interactions based on anarrangement of repetitive hydrogen bonding patterns, for example betweenan oligonucleotide and its complementary oligonucleotide. In oneembodiment, the probe comprises a biological molecule, such as, forexample, a nucleic acid. However, the probe can be any other moleculethat specifically binds to a target. For example, the probe can be aprotein, such as an immunoglobulin, a cell receptor, such as a lectin,or a fragment of any of these, for example, F_(ab) fragment, F_(ab′)fragments, and the like. In another embodiment, the probe can be a cellor particle, such as viral particle, and the target can be a molecule,cell, or other particle that can interact with the cell or particle.

As used herein, a “target” refers to one or more molecules, a particles,or a cells suspected to be present in a sample. For example, a targetcan be a nucleic acid. The target can specifically interact with aparticular probe based on interactions exemplified above. The target canbe detected and/or quantitated in the method or system of the invention.Typically the target is coupled to a “target marker” to allow detectionof the target. The target can comprise naturally occurring or man-mademolecules, and it can be detected in its unaltered state or asaggregates with other species. Examples of targets include antibodies,nucleic acids, receptors, hormones, drugs, metabolites, cofactors,peptides, enzymes, viral particles, cells and the like. In oneembodiment, the target comprises a nucleic acid to be detected in asample. The probe and target are typically members of a specific bindingpair, wherein the members of the pair are known to bind to each other,while binding little or not at all to other nonspecific substances.

The term “sample” is used in its broadest sense. The term includes aspecimen or culture suspected of containing target.

As used herein, the terms “complementary” or “complementarity,” whenused in reference to nucleic acids, specifically, a sequence ofnucleotides such as a probe nucleic acid or a target nucleic acid, referto paired sequences that are able form standard Watson Crick base-pairs.For example, for the sequence “5′-T-G-A-3′,” the complementary sequenceis “3′-A-C-T-5′.” Complementarity can be “partial,” in which only someof the bases of the nucleic acids are matched according to the basepairing rules. Alternatively, there can be “complete” or “total”complementarity between the nucleic acids. The degree of complementaritybetween the nucleic acid strands has effects on the efficiency andstrength of hybridization between the nucleic acid strands.

The term “hybridization” is used in reference to the pairing ofcomplementary nucleic acids. Hybridization and the strength ofhybridization, specifically, the strength of the association between thenucleic acids, is influenced by such factors as the degree ofcomplementarity between the paired nucleic acids, stringency of theconditions involved, the melting temperature (T_(m)) of the formedhybrid, and the G+C/A+T ratio within the nucleic acids.

As used herein, the term “nucleic acid” refers to any of the group ofpolynucleotide compounds having bases derived from purine andpyrimidine. The term “nucleic acid” can be used to refer to individualnucleotides or oligonucleotides, for example, a short chain nucleic acidsequence of at least two nucleotides covalently linked together,typically less than about 500 nucleotides in length, and more typicallyabout 20 to 100 nucleotides in length. The term “nucleic acid” can alsorefer to long sequences of nucleic acid, such as those found in cDNAs orPCR products, for example, sequences that are hundreds or thousands ofnucleotides in length. The exact size of the nucleic acid sequenceaccording to the invention will depend upon many factors, which in turndepend upon the ultimate function or use of the nucleic acid.

Nucleic acids can be prepared using techniques presently available inthe art, such as solid support nucleic acid synthesis, DNA replication,reverse transcription, and the like. Alternately, nucleic acids can beisolated from natural sources. The nucleic acid can be in any suitableform, for example, single stranded, double stranded, or as anucleoprotein. A nucleic acid will generally contain phosphodiesterbonds, although, in some cases, a nucleotide can have an analogousbackbone, for example, a peptide nucleic acid (PNA). Nucleic acidsinclude deoxyribonucleic acid (DNA), for example, complementary DNA(cDNA) ribonucleic acid (RNA), and peptide nucleic acid (PNA). Thenucleic acid can include DNA, both genomic DNA and cDNA, RNA, or bothDNA and RNA, wherein the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides. Furthermore, the nucleic acid caninclude any combination of uracil, adenine, guanine, thymine, cytosineas well as other bases such as inosine, xanthenes, hypoxanthine andother non-standard or artificial bases. PNA is a DNA mimic in which thenative sugar phosphate DNA backbone has been replaced by a polypeptide.

As used herein, “coupling” refers to the direct or indirect attachmentof one moiety to another through the formation of at least one bond,which can include covalent, ionic, coordinative, hydrogen, or Van derWaals bonds, or non-chemical interactions, for example, hydrophobicinteractions. For example, coupling of compound “A” to compound “D” canbe direct and involve the formation of a covalent bond between “A” and“D”, or coupling of compound “A” to compound “D” can be indirect andinvolve the presence of compound “B” and “C” where coordinative bondsexist between “A” and “B”, and “C” and “D”, and a covalent bond existsbetween “B” and “C”. It is understood that according to this descriptionthat two moieties can be coupled to each other by numerous ways. Suchcoupling can include, but is not limited to, specific non-covalentaffinity interations, for example streptavidin: or avidin:biotininteractions and hapten:antibody interactions; hydrophobic interactions;magnetic interactions; polar interactions, for example, “wetting”associations between two polar surfaces or betweenoligonucleotide/polyethylene glycol; formation of a covalent bond, forexample, an amide bond, a disulfide bond, a thioether bond, an etherbond, a carbon-carbon bond; or via other crosslinking agents; or via anacid-labile linker. As used herein, “bonding” refers to the directattachment of two moieties typically through a chemical bond.

“Hydrophilic” and “hydrophobic” are used herein to describe compositionsbroadly as water attracting and water repelling, respectively.Generally, hydrophilic compounds are relatively polar and oftenionizable. Such compounds usually bind water molecules strongly.Hydrophobic compounds are usually relatively non-polar and non-ionizing.“Hydrophobic” refers to materials or surfaces that have a low affinityfor water, are not readily mixed with or wetted by water, and which aregenerally water-repellant. Hydrophobic and hydrophilic are relativeterms and are used herein in the sense that various compositions,liquids and surfaces can be hydrophobic or hydrophilic relative to oneanother.

The present invention provides a method for detecting target in a sampleusing arrays providing a plurality of microparticles having uniqueself-encoding marker/probe pairs. According to the invention, an arrayis provided that includes a set or subset of microparticles havingunique self-encoding marker/probe pairs. Each self-encoding marker/probepair includes a microparticle having a unique self-encoding marker thatcomprises at least one detectable species, and a probe, wherein theself-encoding marker is chosen to correspond with the probe. In oneembodiment, sample suspected to contain a target is treated to couple atarget marker to the target and then the sample containing target-markerlabeled target is applied to the array, so that target marker-labeledtarget, if present in the sample, will hybridize with a probe associatedwith the array. In another embodiment, target marker is coupled to thetarget after binding of the target to the probe. Thereafter, boundtarget and target marker associated with the microparticle having theself-encoding marker/probe pair is detected using a detection scheme.The detection scheme involves detection of both the target marker andthe self-encoding marker. Detection of the self-encoding marker allowsfor determination of the probe, and thus the identity of the boundtarget.

According to the invention, arrays are prepared by randomly disposingand immobilizing microparticles having unique self-encoding marker/probepairs on the substrate wherein an immobilization material is used toimmobilize the microparticles having unique self-encoding marker/probepairs on the substrate. The location and identification of amicroparticle bearing a particular self-encoding marker/probe pair istypically not determined until the detection step. In some embodimentsthe random disposing and immobilization of the microparticles havingunique self-encoding marker/probe pairs can be performed on at least aportion of the substrate that has been patterned with the immobilizationmaterial. A patterned substrate can be used to form multiple arrays,subarrays, or combinations thereof. In one embodiment the microparticlesare immobilized on the surface of the substrate via a polymer that hasbeen disposed on the substrate. In another embodiment the microparticlesare immobilized on the substrate by coupling the microparticles to thesubstrate surface via, for example, a binding pair or a crosslinkingagent. The random disposing and immobilization of sets or subsets ofmicroparticles having unique self-encoding marker/probe pairs canprovide advantages relating to fabrication of arrays, since the preciselocation of a microparticle need not be predetermined. In addition theuse of microparticles provides a greater surface area for probe coupling

The invention contemplates methods for detecting target using the arraysof the invention, methods of making the arrays, and the arraysthemselves. Also contemplated are kits for detecting target in a sample.

Arrays prepared according to the invention are fabricated on a solidsupport, also referred to herein as a “substrate”. Preferably, thesubstrate comprises a integral support. Generally, the term “solidsupport” or “substrate” refers to a material that provides a two- orthree-dimensional surface on which the microparticles of the inventioncan be immobilized. The composition of the solid support can be any sortof suitable material to which the microparticles can be directly orindirectly immobilized. Typically, the microparticles of the inventionare coupled to the “substrate surface” via an immobilization material.The composition of the substrate can vary, depending upon the particularmicroparticles to be immobilized, as well as the desired surfacecharacteristics in areas of the substrate that do not havemicroparticles immobilized on them, as will be discussed herein.

Preferably, the substrate does not interfere with the ability of theprobe to bind target and is not subject to high amounts of non-specificbinding. Suitable materials for the substrate include biological ornonbiological, organic or inorganic materials. Suitable solid substratesinclude, but are not limited to, those made of plastics, ceramic,resins, polysaccharides, silicon and silica-based materials, glass,metals, films, gels, membranes, nylon, natural fibers such as silk, wooland cotton and polymers. Suitable polymers include, but are not limitedto, polystyrene, polyethylene, polyethylene tetraphthalate, polyvinylacetate, polyvinyl chloride, polyacrylonitrile, polymethyl methacrylate(PMMA), butyl rubber, styrenebutadiene rubber, natural rubber,polypropylene, (poly)tetrafluoroethylene, (poly)vinylidenefluoride,polycarbonate, and polymethylpentene. In one embodiment, the substrateis prepared from a material that is suitable for use with a fluorescencedetection device, for example, a fluorescence scanner or a fluorescencemicroscope. In this embodiment, the material for the substratepreferably does not interfere with the detection of the fluorescencesignal associated with the array.

Preferably, the substrate comprises an integral surface forimmobilization of the microparticles. The substrate can be either“substantially flat”, meaning that the surface is substantially planarand has little or no surface configurations, or the substrate can havesurface configurations such as raised portions, surface projections,etched areas, wells, and the like. The surface of the substratepreferably is provided by a single substrate.

The dimensions of the substrate can vary and can be determined by suchfactors, for example, as the dimensions of the array, and the amount ofprobe diversity desired. In some embodiments the substrate can provide asurface for a single array, an array containing multiple arrays, anarray containing subarrays, or an array containing combinations ofmultiple arrays and subarrays. The arrangement of sets or subsets ofrandomly disposed microparticles on the substrate, which can be in theform of multiple arrays or subarrays, can be formed by pre-coating orcoating the substrate with a compound or compounds that facilitate theimmobilization of microparticles on the surface of the substrate,referred to as “immobilization material”. As used herein, the term“pre-coating” or “coating” refers to the process of disposing a compoundon a surface; the compound or compounds can be used for immobilizing themicroparticle on the substrate. In some embodiments the substrate isprecoated with a compound, for example, a polymeric compound, havingphotoreactive groups. Photoreactive groups can be used to couple variousmoieties of the invention to the substrate through the formation ofchemical bonds. Examples of photoreactive groups are described below.

The microparticles of the invention can comprise any three-dimensionalstructure that can be immobilized on a substrate and coupled to theself-encoding marker and probe moieties. Typically the microparticlesare spherular in shape. As used herein “spherular” refers to threedimensional shapes that include, spherical, spheroidal, rounded,globular shapes and the like. The size of the microparticle can be inthe range of about 100 nm to about 100 μm in diameter. In preferredembodiments, the microparticles are of an appropriate size to bedetected as individual particles using the imaging devices describedherein. For example, if the resolution of the imaging device is 5 μm, itis preferable that the microparticles immobilized on the substrate be 10μm or larger.

According to the invention, the microparticle can be fabricated from anysuitable material. Suitable materials include, for example, polymerssuch as poly(methylmethacrylate), polystyrene, polyethylene,polypropylene, polyamide, polyester, polvinylidenedifluoride (PVDF), andthe like; natural polymers such as cellulose, crosslinked agarose,dextran, and collagen; glass, including controlled pore glass (CPG) andsilica (nonporous glass); metals such as gold, steel, silver, aluminum,copper, ferric oxide, and the like; magnetite, and the like. Examples ofuseful microparticles are described, for example, in the “MicroparticleDetection Guide” from Bangs Laboratories, Fishers, Ind.

In some embodiments it is preferable that the microparticles areswellable, and become more porous when placed in an appropriatesolution. As used herein, “swellable” refers to the ability of themicroparticles to expand and become more porous when in an appropriatemedium and can incorporate compounds or particles in a swollen state.Swellable microparticles are useful for impregnating a particle, forexample, a detectable particle, which can be a portion of theself-encoding marker, into the microparticle. Such swellablemicroparticles are typically composed of polystyrene, or copolymers ofpolystyrene, and can be swollen in an organic solvent. Swellablemicroparticles can be useful for incorporating different types ofdetectable material, for example, a magnetic material. In otherembodiments the microparticles can be impregnated with a combination ofdifferent types of detectable material.

Microparticles can also be obtained commercially from, for example,Bangs Laboratories (Fishers, Ind.), Polysciences (Germany), MolecularProbes (Eugene, Oreg.), Duke Scientific Corporation (Palo Alto, Calif.),Seradyn Particle Technology (Indianapolis, Ind.), and Dynal Biotech(Oslo, Norway). Commercially available microparticles can be modifiedfurther to provide the desired self-encoding marker and probe moietieson the microparticle.

The microparticles of the invention can possess one or more desirableproperties, such as dimensional stability, optical properties, forexample, size and color. The microparticles can also be chosen toprovide additional desirable attributes, such as a satisfactory density,for example, a density greater than water or other solvent used inpreparation of the array, or properties that allow the microparticle tobe “self-encoded.”

As used herein “detectable species” refers to a detectable moiety thatcan be associated with a microparticle. Typically, the self-encodingmarker comprises at least one detectable species.

In one aspect, the microparticles of the invention can be described as“self-encoding.” As used herein, the terms “self-encoding” or“self-encoded” refers to a unique detectable identity associated withthe microparticle that allows one microparticle to be differentiatedfrom another using the detection techniques described herein. The term“self-encoding marker” refers to one detectable species or a combinationof detectable species, which serve to provide the microparticle with aunique identity that can be determined by one or more types of detectiontechniques. A self-encoding marker associated with the microparticleprovides the self-encoded nature of the microparticles. According to theinvention, a particular self-encoding marker serves as an identifier ofa particular probe attached to the microparticle. Self-encoding of themicroparticles allows the user to create unique marker/probe pairs, inwhich the self-encoding marker is associated with a particular probe.

Properties useful for self-encoding of the microparticles include, butare not limited to, fluorescence, size, shape, magnetic susceptibility,electrostatic properties, and the like. The self-encoded nature of eachmicroparticle is typically associated with at least one type ofdetectable species, for example, a fluorescent compound, which isassociated with the microparticle. In some embodiments the self-encodedmature is dependent on a combination of detectable species associatedwith a particular microparticle. For example, a first microparticle canbe loaded with or coupled to a selected dye, such as a fluorescent dyethat has an excitation/emission maxima of 350/440 nm. In this exemplaryembodiment, the first microparticle associated with this fluorescent dye(350/440 nm) is also coupled to a probe nucleic acid sequence that iscomplementary to a nucleic acid for Gene A, wherein the nucleic acid forGene A is suspected of being present in a sample. The microparticleassociated with fluorescent dye (350/440) and the probe nucleic acidsequence for Gene A constitutes microparticle having a uniqueself-encoding marker/probe pair. A second microparticle can be loadedwith or coupled to a different selected dye, such as a fluorescent dyethat has an excitation/emission maxima of 488/520 nm. This secondmicroparticle fluorescent dye (488/520 nm) associated is coupled to aprobe nucleic acid sequence complementary to Gene B, wherein the nucleicacid for Gene B is suspected of being present in a sample. Themicroparticle including the fluorescent dye (488/520 nm) and the nucleicacid sequence that is complementary to a nucleic acid for Gene Bconstitutes a microparticle bearing a different unique self-encodingmarker/probe pair. A plurality of microparticles bearing differentself-encoding markers/probe pairs is generated and combined to form a“set” or “subset” of microparticles. The “set” or “subset” ofmicroparticles is then disposed on a substrate or a portion of asubstrate and on the substrate or a portion of the substrate themicroparticles are randomly located. It will be apparent to one of skillin the art that any number of desired fluorescent dyes can be chosen tocorrespond to specific biomolecules, as desired.

Other useful markers that can be incorporated into or coupled to themicroparticles include phosphors, quantum dots, radioisotopes, forexample, molecules containing ³²P, ³³P, ³⁵S, and fluorescence proteins,for example, the Green Fluorescence Protein.

A plurality of unique self-encoded microparticles can be created by, foreach unique microparticle in the plurality, combining differentself-encoding markers on the same microparticle. For example,microparticles having unique self-encoding marker/probe pairs can becreated by incorporating or coupling the microparticle to two, or morethan two, different fluorophores having different emission maxima.Preferably the emission maxima for the different fluorophores aredistinct or are separable through use of band-pass filters, for example.Microparticles can also be prepared by incorporating or coupling themicroparticle to two, or more than two different types of detectablespecies, for example, coupling the microparticle to a fluorophore andalso to a radioisotope. By preparing microparticles having differentcombinations of detectable species, a vast number of microparticles withunique self-encoding markers can be prepared. This is especially usefulwhen the array provides a substantial number of individually uniqueprobes, thus requiring a substantial number of microparticles withindividually unique self-encoding markers. In yet other embodiments, themicroparticles provided in an array comprise a population ofmicroparticles comprising a single fluorescent dye, as well as apopulation of microparticles comprising more than one fluorescent dye.

A plurality of unique self-encoded microparticles can also be createdby, for each unique microparticle in the plurality, combining detectablespecies on the same microparticle at different concentrations. Inaddition to variation in the emission type, variation in the emissionintensity, as a result of varying the concentration of the detectablespecies, can also provide additional variation to establishing aplurality of self-encoding markers. In one exemplary embodiment, 5 nmolof fluorescent dye A (488/520 nm) and 50 nmol of fluorescent dye B(350/440) are incorporated or coupled to microparticle C. MicroparticleD has 50 nmol of fluorescent dye A (488/520 nm) and 5 nmol offluorescent dye (350/440) incorporated or coupled to it. Althoughmicroparticle C and microparticle D include the same fluorescent dyes,they are detectable from one another because the intensity of emissionsof fluorescent dyes A and B are different for microparticle C andmicroparticle D. Such fluorescent dyes and the intensity of emission ofthese dyes can be detected using imaging devices.

According to the invention, individual self-encoded microparticles canbe distinguished in the array by taking the mean fluorescence intensityminus the background intensity at each emission wavelength for theselected dye or dyes. In the case of a microparticle encoded withmultiple dyes, the mean fluorescence intensity minus the backgroundintensity at each emission wavelength is taken and then divided by thefluorescence intensity of the alternate dye at its wavelength maxima todetermine the signature of the particular dye ratio used to encode themicroparticle. Unique sets of encoding dyes can be determined accordingto the formula (n!/p!) (n−p)!, where n is the number of dyes and p isthe number of combinations (Michael et. al., (1998) Anal. Chem.,70:1242) Examples of suitable fluorescent dyes include, but are notlimited to, Indodicarbocyanine, Texas Red cadaverine, fluorescein,Oregon Green 488 (2′,7′-difluorofluorescein), BODIPY-based dyes, that isdyes containing 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, Alexa 532,Cy 3, Cy5, tetramethylrhodamine, and other rhodamine derivatives.Microparticles can be coupled to or impregnated with the desiredfluorescent dye or combination of fluorescent dyes to achieve thedesired fluorescent properties that defines the self-encoding marker.

In another embodiment, the microparticles of the current invention canbe self-encoded by embedding nanocrystals on or within themicroparticles that confer particular optical properties to themicroparticles. These nanocrystals can include quantum dots. Forexample, the self-encoding of microparticles can be achieved byembedding different sized quantum dots into microparticles at distinctratios. These quantum dots can be composed of compounds such as zincsulfide-capped cadmium selenide, indium aresenide, cadmium telluride,and the like. The optical properties of these quantum dots typicallyinclude a high luminescence and a size tunable emission and simultaneousexcitation.

Different fluorescence emission from quantum dots is typically due tothe size of the quantum dot itself. Fluorescence emission wavelength canbe changed by altering the size of the quantum dot, and a singleexcitation wavelength can be used for the simultaneous excitation ofdifferent-sized quantum dots.

Quantum dots can be incorporated into and spatially separated from eachother in the microparticles. Quantum dots that are sufficiently spacedfrom one another typically do not undergo fluorescence resonance energytransfer. Quantum dots can also be incorporated into microparticles atprecisely controlled ratios, allowing one to generate a plurality ofmulticolor microparticles each with a different combination and amountof quantum dots.

Microparticles formed by emulsion polymerization of styrene,divinylbenzene, acrylic acid, and like materials are suitable forincorporating quantum dots. Preferably, microparticles composed of amaterial which allows the separation of incorporated quantum dots anddoes not allow the incorporation of larger particles and aggregates arepreferred. In the preparation of microparticles including quantum dots,typically the largest size quantum, dots are loaded first and thesmallest quantum dots are loaded last.

Microparticles containing quantum dots can also be sealed using apolymer material, for example a polysilane layer. A polymer material canstabilize the optical properties of the quantum dots, prevent exposureto reagents used when probing a sample, and also protect againsttemperature variations.

In one embodiment, the microparticles are self-encoded by incorporatinga selected fluorescent dye into the microparticles. For example, themicroparticles can be soaked in organic solvents containing a selecteddye or dyes. The solvent swells the polymeric microparticles and allowsthe dyes to penetrate into the microparticles' cores. Excess solvent isthen removed, for example, by vacuum filtration, entrapping the dyes inthe interior regions of the microparticles. In one example,poly(methylsytrene)-divinyl benzene microparticles are rinsed indimethylformamide. A solution containing the selected dye or dyes indimethylformamide is then added to the microparticles, and themicroparticles and solution are incubated with agitation for 24 hours.Excess dye is removed from the suspension by vacuum filtration usingmembrane filters, such as those provided by Millipore Company (Bedford,Mass.). The filtered microparticles are then sonicated and washed bycentrifugation in distilled water containing 0.01% Tween 20 to removeresidual dye on the outside of the microparticles.

Alternatively, fluorescent microparticles can be obtained commercially,for example, from Molecular Probes, Inc. (Eugene, Oreg.). MolecularProbes provides a variety of fluorescent microparticles under theproduct name FluoSpheres™, which are polystyrene microparticles loadedwith various proprietary dyes with excitation and emission wavelengthsranging from near ultraviolet to the near infrared. These FluoSpheres™can also be prepared with intensities lower than those of the regularselection, which can be desirable in some multicolor applications. Othercommercial sources of fluorescent microparticles, for example, fromLuminex Corporation (Austin, Tex.) can be used in accordance with theinvention. Luminex provides a number of internally color-codedmicroparticles using a blend of different fluorescent intensities of twodyes. These microparticles can be provided with an activated surface,such as a carboxylated coating or avidin coating.

In some embodiments, self-encoding of the microparticle is accomplishedby the size or shape of the microparticle. Differences in sizes can bevisualized using equipment such as an image analyzer or an arrayscanner, for example. In order to differentiate microparticles accordingto size the difference between the sizes of microparticles is preferablynot less than 50%, based on the diameter of the microparticles. Formicroparticles that are larger, for example approximately 50 μm, it issufficient that the size difference is approximately 20%. Microparticlescan also be distinguished according to shapes, for example, spherular ormisshapen shapes. As used herein, misshapen microparticles display ashape that is discernable from a spherular shape and can be visualizedusing equipment such as, for example, an image analyzer, an arrayscanner, or a microscope. The shape of misshaped microparticles can be,for example, elongated or rod-like. In these embodiments, theself-encoding marker is a physical characteristic of the microparticleitself, such as the size or shape of the microparticle.

In some embodiments, self-encoding of the microparticle can be providedby magnetic or electrostatic properties of the microparticle. Magneticmicroparticles are commercially available from, for example, DynalBiotech (Oslo, Norway). Detection of electrostatic microparticles can beaccomplished, for example, by placing the microparticle on a chargedsurface or in an electric field. One useful application is to placeelectrostatic microparticles on an array containing at least oneelectrode. A charge can be applied to the electrode, and depending onthe charge associated with the microparticle, the microparticle will beattracted or repelled from the charge. Magnetic microparticles can bedetected by placing them in a magnetized area. Magnetic properties ofmicroparticles can be measured on assay surfaces adapted or use withreaders for magnetic tapes, disks, and the like.

Visualization of the fluorescent dyes can be accomplished using anysuitable visualization technique known in the art. Fluorescence imagingcan be made using a modified epifluorescence microscope or afluorescence confocal microscope. Suitable microscopes include, forexample, an Olympus BX60 (Tokyo, Japan) or other similar microscopes.Fluorescence images from microscopy images can be analyzed forfluorescence intensity using computer software. Commercially availablemicroscopy analysis software, for example, Image—Pro Plus (version 4.0)(Media Cybernetics, L.P., Silver Spring, Md.), can be used to define andcount fluorescent signals automatically with optical detection systems.Alternatively, the method of fluorescence scanning can be used tovisualize microparticles having self-encoded markers. Fluorescencescanners such as the Scan Array 5000 (GSI Lumonics, Billerica, Mass.) orAxon GenePix 4000A (Foster City, Calif.), which have resolution ofapproximately 5 μm, can be used for visualization.

It will be readily apparent to one of skill in the art, upon review ofthis disclosure, that an array can include a plurality of microparticleshaving combinations of the above-described detectable species. Forexample, a single array can utilize two or more specific types ofdetectable species, selected from fluorescent dyes, physicalcharacteristics, such as size or shape, and magnetic or electrostaticproperties. The choice of a particular combination of detectable speciescan be determined based upon the visualization techniques and equipmentavailable to the user.

In some embodiments it is useful to generate a “set” of self-encodedmicroparticles. The members of a “set” of self-encoded microparticlesare preferably readily distinguished from one another using thedetection techniques as described herein. A set of self-encodedmicroparticles can contain approximately 2-10,000 different self-encodedmicroparticles but preferably contains approximately 50-200 differentself-encoded microparticles. A set of different self-encodedmicroparticles can be used to form multiple arrays on a single substrateor subarrays on a single substrate by the methods discussed below. Insome embodiments the set of microparticles can be coupled to “subsets”of probe molecules.

The surface of the microparticles can, in some embodiments, befunctionalized to provide “reactive groups” for coupling one or moreprobes to the microparticle, for coupling one or more detectablemoieties to the microparticle, for coupling the microparticles to eachother, or for coupling the microparticles to the immobilization materialor the substrate. Suitable reactive groups can be chosen according tothe nature of the moiety that is to be attached to the microparticle.Examples of suitable reactive groups include, but are not limited to,carboxylic acids, sulfonic acids, phosphoric acids, phosphonic acids,aldehyde groups, amine groups, thiol groups, thiol-reactive groups,expoxide groups, and the like. For example, carboxylate-functionalizedmicroparticles can be used for covalent coupling of proteins and otheramine-containing molecules using water-soluble carbodiimide reagents.Aldehyde-functionalized microparticles can be used to couple themicroparticles to proteins and other amines under mild conditions.Amine-functionalized microparticles can be used to couple themicroparticles to a variety of amine-reactive moieties, such assuccinimidyl esters and isothiocyanates of haptens and drugs, orcarboxylic acids of proteins. In another embodiment, sulfate-modifiedmicroparticles can be used when the user desires to passively absorb aprotein such as bovine serum albumin (BSA), IgG, avidin, streptavidin,and the like. In another embodiment, the reactive groups can includesuch binding groups as biotin, avidin, streptavidin, protein A, and thelike. Functionalized microparticles are also commercially available froma number of commercial sources, including Molecular Probes, Inc.(Eugene, Oreg.).

In some embodiments, microparticles can be coupled to other moietiesthrough use of crosslinking agents. Commercially available crosslinkingagents obtained from, for example, Pierce Chemical Company (Rockford,Ill.) can be used to link microparticles together via, for example,amine groups of the proteins. Useful crosslinking agents includehomobifunctional and heterobifunctional crosslinkers. Two non-limitingexamples of crosslinking agents that can be used on coatedmicroparticles are di-succinimidyl suberate and 1,4-bis-maleimidobutane.

According to the invention, an array comprises a substrate and aplurality of microparticles associated with unique self-encodingmarker/probe pairs immobilized on the substrate. As described herein,the probe comprises a moiety which can recognize a particular target,such as, for example, a nucleic acid with a unique sequence. In otherembodiments the probe comprises a particle, a cell, or a portion of acell. Given the teachings herein, one of skill in the art can select adesired probe, or set of desired probes, for fabrication of an array.Typically, the probe comprises a biological molecule.

In one embodiment the probe is a nucleic acid and the array includes aplurality of unique self-encoded microparticles coupled to a pluralityof different nucleic acids defined by having different nucleic acidsequences. The nucleic acid probes coupled to the microparticles can beof any length but are preferably at least 6 nucleotides in length. Morepreferably the nucleic acid probes are between 8 and 200 nucleotides inlength and most preferably the nucleic acids probes are between 12 and50 nucleotides in length.

In another embodiment the probe can be a protein molecule, or a complexof protein molecules, and the array includes a plurality ofmicroparticles having unique self-encoded markers, the microparticlesalso coupled to a plurality of different protein molecules, or a complexof protein molecules, each having an affinity for a target which can bepresent in a sample. The protein molecule can be, for example, anantibody that specifically recognizes a target or portion of a target,if present in a sample. The array can therefore comprise a plurality ofself-encoded microparticles coupled to different antibody molecules,each with an affinity for a particular target, which may be present in asample. Protein probes can include but are not limited to, for example,cell surface receptors, cell surface ligands, and intracellular proteinsthat interact specifically with another molecule.

In some embodiments a self-encoding microparticle “set” can be prepared,as described above, and individual members of the “set” can be coupledto unique probe molecules. In some embodiments, if the number of probemolecules greatly exceeds the number of individual self-encodingmicroparticles in the set, “subsets” of self-encoding marker/probe pairscan be generated. The following example provides a description of thepreparation of subsets of microparticles having self-encodingmarker/probe pairs. In an exemplary embodiment, a set of self-encodingmicroparticles is prepared which includes 100 unique self-encodingmicroparticle batches, and a set of probe molecules is prepared whichincludes 5000 unique probe nucleic acid molecules. The 5000 probemolecules are divided into 50 subsets of probes, each subset containing100 probes. One subset of microparticles having self-encodingmarker/probe pairs is prepared by individually coupling one member ofthe self-encoded microparticle set with one member of a probe subset.The set of self-encoded microparticles is used repeatedly to generatethe 50 subsets of microparticles having self-encoding marker/probepairs. In fabrication of an array, each of the 50 subsets containingmicroparticles having self-encoding marker/probe pairs can be disposedon the substrate at predetermined locations and are kept spatiallyseparated. Detection of a microparticle having a particular self-encodedmarker/probe pair is based on both the features of the detectablespecie(s) on the microparticle and also the location of the subset onthe substrate.

In some embodiments, it can be useful to group microparticles havingself-encoding marker/probe pairs into subsets based on the commonattribute of the probe molecule. For example, it may be useful to createsubsets of microparticles having self-encoding marker/probe pairs basedon tissue-specific or disease-specific expression of the target thatbinds expressly to the probe. Subsets can also be based on the type orclass of molecule reactive within a probe, for example, transcriptionalfactors, such as repressors or activators, or cell surface factors, suchas receptors.

As indicated, a microparticle with a unique self-encoding marker iscoupled to a unique probe. Typically, an individual microparticle iscoupled with a plurality of identical probe molecules. By providingmultiple copies of the same probe molecule on a single microparticle,the sensitivity of the array can be increased. For example, followingbinding of the target to the probe, a higher signal to noise ratio canbe achieved.

The number of probe molecules provided on each individual microparticlecan be adjusted by the user to achieve the desired effect. The densityof probe molecules, for example oligonucleotide or protein probemolecules, on a microparticle can be in the range of 1-260,000 probemolecules per 1 μm diameter microparticle. Typically, 40,000-50,000probe molecules are immobilized per 1 μm diameter microparticle.Accordingly, the amount of probe molecules on the microparticle can alsobe dependent on the size of the microparticle used. However, dependingon microparticle source and preparation, for example, the amount ofstreptavidin bound to a particular microparticle preparation, the amountof probe molecules coupled to the microparticles may vary. According tothe method of the invention the density of probe molecules present onthe array surface can be controlled with greater precision as comparedto conventional methods of array preparation.

Probe molecules are typically coupled to the microparticles prior todeposition and attachment of the microparticles to the substrate. Forexample, the probe can be coupled to the microparticle in a suitableliquid media, such as phosphate buffered saline. Coupling of the probeto the microparticle prior to deposition of the microparticle canprovide benefits in array preparation. For example, probes can becoupled to the microparticles at a higher density as compared tocoupling of the probe to a conventional substrate with a flat surface.This can be accomplished using any of the coupling techniques discussedherein. Once the microparticles have been coupled with the desiredamount and type of probe, these prepared microparticles can then beapplied to the substrate and coupled thereto. Preferably, coupling ofthe microparticles prepared in this way does not interfere with theability of the probe to bind with appropriate targets present in asample. Coupling of probes to microparticles in solution is alsogenerally more efficient than the coupling of probes to a conventionalsubstrate with a flat surface, resulting in a low loss of probe duringthe coupling procedure. In addition, coupling of a probe to amicroparticle in solution generally allows for more variability in thecoupling process. In situations when the process of coupling of a proberequires particular conditions, for example the stirring of a probe insolution to allow coupling of the probe or proper presentation of thecoupled probe, the use of microparticles, which can be mixed or agitatedin solution, can allow these particular coupling conditions to be met.

According to the invention, probes can be coupled to the microparticlesin any suitable manner. For example, the microparticles can be providedwith reactive groups on the surface, as described above. Depending uponthe reactive groups selected and the desired probe, the probe may or maynot be modified prior to attachment to the microparticle.

Coupling of the probe to the microparticle can be accomplished byproviding reactive groups on the microparticle, the probe, or both. Somereactive groups are discussed above for modification of themicroparticles.

In some embodiments, the probe can be modified prior to coupling withthe microparticle. For example, nucleic acids can be coupled to onemember of a binding pair, and the microparticles coupled to the othermember of the binding pair. Suitable binding pairs include but are notlimited to avidin:biotin, streptavidin:biotin, and antibody:hapten.Examples of antibody hapten binding pairs include anti-digoxigeninAb:digoxigenin or anti-trinitrophenyl Ab:trinitrophenyl. For example, anucleic acid can be biotinylated, for example, using enzymaticincorporation of biotinylated nucleotides, or by cross-linking thebiotin to the nucleic acid using methods known in the art. Biotinylatednucleic acid can then be coupled with streptavidin provided on thesurface of the microparticles.

Nucleic acids can be modified in a variety of ways to afford coupling tothe microparticles. For example, nucleic acid can be modified to providea reactive moiety at the 3′ or 5′ end. Alternatively, nucleic acid canbe synthesized with a modified base. In addition, modification of thesugar moiety of a nucleotide at positions other than the 3′ and 5′position is possible through conventional methods. Also, nucleic acidbases can be modified, for example, by using N7- or N9-deazapurinenucleosides or by modification of C-5 of dT with a linker arm, forexample, as described in F. Eckstein, ed., “Oligonucleotides andAnalogues: A Practical Approach,” IRL Press (1991). Alternatively,backbone-modified nucleic acids, such as phosphoramidate DNA, can beused so that a reactive group can be attached to the nitrogen centerprovided by the modified phosphate backbone.

Preferably, the modification of a probe, such as a nucleic acid, doesnot substantially impair the ability of the probe or nucleic acid tohybridize to its complement. In the case of nucleic acid, modificationshould preferably avoid substantially modifying the functionalities ofthe nucleic acid that are responsible for Watson-Crick base pairing.

In one embodiment, one or more photoreactive groups are randomlyattached to the probe nucleic acid, for example along the backbone or ateither their 3′ or 5′ ends. For example, the bases present on thenucleotides making up the nucleic acid possess numerous reactive groupsthat can be derivatized using a heterobifunctional photoreactivecompound having both a photoreactive group and a thermochemicallyreactive group suitable for coupling to the bases. In this embodiment,thermochemically reactive groups, as described herein, can be used tocouple the photoreactive group to the probe nucleic acid via reactivegroups present on the nucleic acid. This approach typically results in arelatively nonselective derivatization of the nucleic acid, both interms of the location along the backbone, as well as the number ofphotoreactive groups per nucleic acid molecule.

In an alternative embodiment, the oligonucleotide can be synthesized toincorporate chemically reactive groups at specific sites of theoligonucleotide, for example, along the backbone or at the 3′ or 5′ends. For example, commercially available reagents or solid supports areavailable that permit the incorporation of amine groups at any of theselocations in the oligonucleotide. These amine groups are then reactedwith a photoreactive compound that includes a thermochemically reactivegroup, as described herein, resulting in formation of an amide bondbetween the photoreactive group and the oligonucleotide. Otherelectrophilic and nucleophilic species can provide similar couplingtechniques.

In another embodiment, one or more of the nucleotide building blockstypically used in oligonucleotide synthesis can themselves bederivatized with a reagent containing a photoreactive group byattachment of the reagent to one of the reactive functionalities presenton the base residue of the nucleotide. The resulting derivatizednucleotide reagent can be used in an automated synthesizer, underconventional reaction conditions, in order to incorporate thephotoreactive group at designated points along the chain or at eitherend of the oligonucleotide. In addition, commercially availablenon-nucleotide reagents, used for incorporation of chemically reactivegroups, can be reacted with the photoreactive compound to incorporatethe photoreactive group, after which they can be used in the automatedsynthesizer to prepare the photoactivatable nucleic acids.

A variety of reagents are available for use in modifying nucleic acids.In another embodiment, photoderivatized nucleotides can be synthesizedand incorporated into nucleic acids using enzymatic techniques. Forexample, a variety of reagents are available that can be used to labelnucleic acids with biotin, fluorescein and digoxigenin (DIG). A nucleicacid can be labeled with a photoreactive dideoxyribonucleotide ordeoxyribonucleotide, using a terminal transferase, in order to provideeither single or multiple photoreactive groups at the 3′ end of thenucleic acid. For example, a DIG-labeling kit called “DIG-High Prime”for use in random primed labeling of DNA with DIG-11-UTP is alsoavailable. “Biotin High Prime”, Boehringer-Mannheim and“Fluorescein-High-Prime” products are also available. In a similarfashion, DNA can be random-primed with a photoreactivedeoxyribonucleotide using the Klenow enzyme.

DNA Polymerase I enzyme can also be used to incorporate photoreactivegroups into an oligonucleotide. By including photoreactivedeoxyribonucleotides in the mixture of deoxynucleotide triphosphates(dNTPs), the resulting polymerized product will contain one or morephotoreactive groups along its length. In addition, during polymerasechain reaction (PCR), a photoreactive deoxyribonucleotide can beincluded in the mixture of dNTPs for the labeling of amplificationproducts. It is also possible to incorporate a photoribonucleotide intoRNA, for example, by the use of an RNA polymerase such as SP6 or T7, andstandard transcription protocols.

Alternatively, polypeptides, for example proteins, can be passivelyabsorbed onto microparticles and then optionally crosslinked to themicroparticles. Polypeptides are preferably absorbed onto themicroparticles under conditions that promote the greatest interaction ofhydrophobic portions of the polypeptide and the microparticle.

The array is generally fabricated by disposing a plurality ofmicroparticles having unique self-encoding marker/probe pairs on asubstrate and allowing the microparticles to become immobilized on thesubstrate. Immobilization of the microparticles can be accomplished byproviding immobilization material which can couple the microparticles tothe surface of the substrate.

As used herein, “immobilization material” collectively refers tocompounds that are used to immobilize the microparticles to the surfaceof the substrate. In some embodiments the immobilization material can bea single compound, for example, a crosslinking agent or a polymericmaterial, which is in direct contact with both the surface of thesubstrate and the microparticle. In other embodiments the immobilizationmaterial can be at least two compounds, for example, two members of abinding pair. When two or more compounds are included in theimmobilization material, these compounds can interact in series to linkthe microparticle to the surface of the substrate. In a preferredembodiment, a reactive immobilization material (RIM) is used forcoupling the substrate to the microparticles. Reactive immobilizationmaterial can include chemically reactive groups, for examplephotoreactive groups, or thermally reactive groups. These reactivegroups are typically responsive to an applied agent, such as light orheat. Activation of these groups can be used to link the treatableimmobilization material to the substrate, microparticle, or otherdesired moieties.

In one embodiment, the substrate can be pre-coated with an organosilanematerial. Pre-coating with an organosilane material can be useful inproviding a surface of the substrate that can form bonds with otherimmobilization material. In this embodiment, the substrate is cleaned,pretreated or cleaned and pretreated prior to attachment of themicroparticles. The substrate (for example, a soda lime glass microscopeslide) is silane treated by dipping it in a mixture of 1%p-tolydimethylchlorosilane (T-silane) and 1% N-decyldimethylchlorosilane(D-silane, United Chemical Technologies, Bristol, Pa.) in acetone, for 1minute. After air drying, the slides are cured in an oven at 120° C. forone hour. The slides are then washed with acetone followed by dipping indistilled water. The slides are further dried in an oven for 5-10minutes. Other pretreatment or washing steps will be apparent uponreview of this disclosure. Optionally, portions of the substrate can bepre-treated with compounds to develop areas having different surfaceproperties, for example, different regions of hydrophobicity orhydrophilicity.

In other embodiments, pretreatment of the substrate is not required, aswill be apparent upon review of this disclosure. In these embodimentsthe substrate can be directly pre-coated with a compound, such as apolymeric compound.

In another embodiment the microparticles can be immobilized on asubstrate by use of a polymer. In this embodiment the substrate iscoated with a suitable polymer prior to or during the step of disposingthe microparticles. Suitable polymers can be synthetic polymers whichcan include polyacrylamide, polymethacrylamide, polyvinylpyrrolidone,polyacrylic acid, polyethylene glycol, polyvinyl alcohol, andpoly(HEMA), copolymers thereof, or any combination of polymers andcopolymers. Natural polymers can also be used and includepolysaccharides, for example, polydextrans, glycosaminoglycans, forexample, hyaluronic acid, and polypeptides, for example, solubleproteins such as albumin and avidin, and combinations of these naturalpolymers. Combinations of natural and synthetic compounds can also beused. The polymers and copolymers as described can also be derivitizedwith a reactive group, for example a thermally reactive group or aphotoreactive group.

In a preferred embodiment, the substrate is coated with a polymer orcopolymer containing at least one photoreactive group, herein referredto as a ‘photoreactive polymer’. Such photoreactive polymers can beprepared by the polymerization of monomers functionalized withphotoreactive groups. Alternatively, the photoreactive polymer can beformed by the polymerization of monomers functionalized withphotoreactive groups and other non-functionalized monomers, therebyforming photoactivatible copolymers.

According to this embodiment, photoreactive groups can be provided on apolymer. As used herein, a “photoreactive polymer” can include one ormore “photoreactive groups.” A “photoreactive group” includes one ormore reactive moieties that respond to a specific applied externalenergy source, such as radiation, to undergo active species generation,for example, active species such as nitrenes, carbenes and excitedketone states, with resultant covalent bonding to an adjacent targetedchemical structure. Examples of such photoreactive groups are describedin U.S. Pat. No. 5,002,582 (Guire et al., commonly owned by the assigneeof the present invention), the disclosure of which is incorporatedherein in its entirety. Photoreactive groups can be chosen to beresponsive to various portions of the electromagnetic spectrum,typically ultraviolet, visible or infrared portions of the spectrum.“Irradiation” refers to the application of electromagnetic radiation toa surface.

Photoreactive aryl ketones are preferred photoreactive groups on thephotoreactive polymer, and can be, for example, acetophenone,benzophenone, anthraquinone, anthrone, and anthrone-like heterocycles(i.e., heterocyclic analogs of anthrone such as those having N, O, or Sin the 10-position), or their substituted (e.g., ring substituted)derivatives. Examples of preferred aryl ketones include heterocyclicderivatives of anthrone, including acridone, xanthone and thioxanthone,and their ring substituted derivatives. Particularly preferred arethioxanthone, and its derivatives, having excitation wavelengths greaterthan about 360 nm.

The azides are also a suitable class of photoreactive groups on thephotoreactive polymer and include arylazides (C₆R₅N₃) such as phenylazide and particularly 4-fluoro-3-nitrophenyl azide, acyl azides(—CO—N₃) such as ethyl azidoformate, phenyl azidoformate, sulfonylazides (—SO₂—N₃) such as benzensulfonyl azide, and phosphoryl azides(RO)₂PON₃ such as diphenyl phosphoryl azide and diethyl phosphorylazide.

Diazo compounds constitute another suitable class of photoreactivegroups on the photoreactive polymers and include diazoalkanes (—CHN₂)such as diazomethane and diphenyldiazomethane, diazoketones (—CO—CHN₂)such as diazoacetophenone and 1-trifluoromethyl-1-diazo-2-pentanone,diazoacetates (—O—CO—CHN₂) such as t-butyl diazoacetate and phenyldiazoacetate, and beta-keto-alpha-diazoacetates (—CO—CN₂—CO—O—) such as3-trifluoromethyl-3-phenyldiazirine, and ketenes (—CH═C═O) such asketene and diphenylketene.

Exemplary photoreactive groups are shown in Table 1. TABLE 1Photoreactive Group Bond Formed aryl azides Amine acyl azides AmideAzidoformates Carbamate sulfonyl azides Sulfonamide phosphoryl azidesPhosphoramide Diazoalkanes new C—C bond Diazoketones new C—C bond andketone Diazoacetates new C—C bond and esterbeta-keto-alpha-diazoacetates new C—C bond and beta-ketoester aliphaticazo new C—C bond Diazirines new C—C bond Ketenes new C—C bondphotoactivated ketones new C—C bond and alcohol

The photoreactive polymer can, in some embodiments, comprise aphotoreactive copolymer. The polymer or copolymer can have, for example,a polyacrylamide backbone or be a polyethylene oxide-based polymer orcopolymer. Examples of a photoreactive polymers and copolymers include acopolymer of vinylpyrrolidone andN-[3-(4-Benzoylbenzamido)propyl]methacrylamide (BBA-APMA); anotherexample is a copolymer of acrylamide and BBA-APMA. Other examples ofphotoreactive groups and their attachment to polymers can be found inthis description. It is envisioned that a wide variety of photoreactivepolymers and photoreactive copolymers can be synthesized according tothe materials described herein which allow the immobilization ofmicroparticles on the surface of the substrate.

The photoreactive groups of the photoreactive polymer can allow theformation of a covalent bond between the substrate and the photoreactivepolymer thereby binding the polymer to the surface of the substrate. Thephotoreactive groups of the photoreactive polymer can also serve tocrosslink to proximal polymeric strands together, allowing the formationof a network of covalently crosslinked polymeric strands that serve toimmobilize microsparticles. In some embodiments, a nonphotoreactivecrosslinking agent can be used to promote the formation of crosslinkedpolymeric strands. Use of a crosslinking reagent, for examplebis-acrylamide, can depend on the location and number of photoreactivegroups that are present on the polymeric strand.

In forming the polymeric matrix, the polymer can be applied to thesubstrate and then treated to crosslink the polymers. In one embodiment,a slurry including microparticles and polymer is disposed on thesubstrate and then the polymer is treated to crosslink the polymer, forexample, by activation of reactive groups provided by the polymer.

Different concentrations of polymer can be present in the slurry butgenerally the concentration shouls be great enough to allow forimmobilization of the microparticles. The concentration of the polymercan also depend on the size of the microparticles used. For example, theconcentration of polymer is at least 0.625 mg/mL for a 1.0 μmmicroparticle in order to stably entrap the microparticle in thepolymeric matrix.

In other embodiments the microparticles can be coupled to the surface ofthe substrate using members of a binding pair (for example, bindingpartner A and binding partner B). One member of the binding pair can bebound to a desired location on the substrate and the other member of thebiding pair can be bound to the microparticle. The interaction betweenthe binding pair member and the substrate or the microparticle can beaccomplished by covalent, ionic, coordinative, or hydrogen bonding orcombinations thereof. Non-limiting examples of partners (for example,Partner A and Partner B) in binding pairs are provided in Table 2: TABLE2 Partner A of Binding Pair Partner B of Binding Pair antigen or haptenanti-antigen or anti-hapten antibody or antibody fragment Anti-antigenor anti-hapten antigen or hapten antibody or antibody fragment Hormonehormone receptor hormone receptor Hormone avidin, streptavidin, Biotinor biotinylated compound neutravidin, or avidin- or complex containingcompound or complex biotin or biotinylated compound avidin,streptavidin, neutravidin, or complex or avidin-containing compound orcomplex protein A or G Immunoglobulin Immunoglobulin protein A or GEnzyme enzyme cofactor or inhibitor enzyme cofactor or inhibitor Enzymelectin or organoborate Carbohydrate Carbohydrate Lectin or organoborate

Another method for coupling moieties of the invention is through acombination of chemical and affinity interactions, herein referred to as“chemi-affinity” interactions, as described by Chumura et al. (2001,Proc. Natl. Acad. Sci., 98:8480). Binding pairs can be engineered thathave high binding specificity and a neglible dissociation constant byfunctionalizing each member of the binding pair, near the affinitybinding sites of the pair, with groups that will react to form acovalent bond. For example, the substituents of each functionalizedmember can react, for example by Michael addition or nucleophilicsubstitution, to form a covalent bond, for example a thioether bond.Antigen:anti-antigen antibody pairs, complementary nucleic acids, andcarbohydrate:lectin pairs are example of binding pairs that can befunctionalized to provide chemi-affinity binding pairs.

In other embodiments, the array can be fabricated by disposing, andoptionally treating the immobilization material on the substrate so thatsets or subsets of microparticles having unique self-encodingmarker/probe pairs can be randomly disposed on the substrate.

In one embodiment, all or a portion of the surface of the substrate iscoated with a reactive immobilization material and the reactiveimmobilization material is treated to bind it to the substrate. Forexample, a photoreactive polymer is coated onto the substrate andirradiated to form covalent bonds between the substrate and thephotoreactive polymer. In another example, a member of a binding pair,for example biotin, is disposed on the surface of a substrate that hasbeen modified to present chemical groups reactive towards the biotin andwhich binds biotin to the surface. Microparticles coupled tostreptavidin can be immobilized on this biotin-coupled surface. Examplesof members of binding pairs that can be used in this embodiment areshown in Table 2. In yet another example, a crosslinking agent can becoated on a substrate and used to couple the microparticle to thesubstrate. In some embodiments the concentration of the immobilizationmaterial can also be adjusted to provide a desired amount ofimmobilization material on the surface of the substrate. This, in turn,can be useful for providing an array surface where the amount of boundimmobilization material controls the spacing of the microparticles. Inthese embodiments, the microparticles can be disposed on a substratethat has been coated with the immobilization material and themicroparticles become immobilized randomly on the substrate via theimmobilization material.

In other embodiments, a pattern of immobilization material can be formedon the substrate allowing microparticles to be coupled to the substrateat locations where immobilization material is present. Forming of apattern of immobilization material on a substrate can be useful forcreating a substrate that contains multiple arrays or subarrays ofmicroparticles. The pattern of immobilization material formed on thesubstrate can be of any shape or size. As shown in FIG. 1, theimmobilization material can be patterned on the substrate in multiplesquares on the surface of the substrate, thereby forming “patches” ofimmobilization material 110. The microparticles become immobilized on orwithin the patches when the microparticles are disposed on thesubstrate.

The pattern can be formed on the surface by a variety of methods. In oneembodiment, as illustrated in FIG. 1, a substrate 102 can be coated withan immobilization material (IM) having photoreactive groups therebyforming photoreactive IM-coated substrate 104. A mask 106 is placed overthe photoreactive IM-coated substrate 104 and then exposed to an lightsource to activate the photoreactive groups and bind the immobilizationmaterial to areas on the substrate 102 surface that are not protected bythe mask 106. Unbound photoreactive IM can be removed by, for example, awashing step, resulting in a patterned substrate 108 containing patches110 of bound immobilization material. In this embodiment, a preferredphotoreactive immobilization material is a photoreactive polymer, forexample, a copolymer of vinylpyrrolidone andN-[3-(4-Benzoylbenzamido)propyl]methacrylamide (BBA-APMA); or acopolymer of acrylamide and BBA-APMA.

In another embodiment as illustrated in FIG. 2, a mask 106 is placedover a substrate 102 which is then sprayed with an immobilizationmaterial (IM) reactive with the surface of the substrate 104. The mask106 can be removed leaving a pattern of immobilization materialresulting in a patterned substrate 118 containing patches 120 of boundimmobilization material. In some cases, the patterned substrate 118 canbe treated if the immobilization material contains reactive groups.

Patterned substrates can be used for the fabrication of arrayscontaining subarrays or multiple arrays. In one embodiment, asillustrated in FIG. 3, the fabrication of an array having subarrays isdemonstrated. FIG. 3 a depicts the preparation of a self-encodedmicroparticle set by coupling different combinations of detectablespecies to microparticles. The self-encoded microparticle set of 3 acontains 100 different self-encoding microparticles; however the set cancontain any number of different self-encoding microparticles as definedby the number of different self-encoding combinations. FIG. 3 b and 3 cdepict two different “subsets” of probes. Each subset typically containsa different group of probes, however redundancy in probe presencebetween subsets can occur. In this example, self-encoded microparticlesfrom FIG. 3 a are coupled to probes from FIG. 3 b to create a subset ofmicroparticles having unique self-encoding marker/probe pairs as shownin FIG. 3 d. Another subset of unique self-encoded marker/probe pairs iscreated by coupling the microparticle set of FIG. 3 a with the probesubset of FIG. 3 c. Any number of subsets of microparticles withself-encoding marker/probe combinations can be prepared. Typically, thenumber of subsets is defined by the number of unique probe molecules tobe displayed on the array.

Upon preparation of subset(s) of microparticles having self-encodingmarker/probe pairs, the members of the set can be mixed together fordisposing on a patterned substrate 128. Disposing of the subsets ofmicroparticles can be performed by any acceptable method, for example,by pin printing or by application with a needle.

In this example, the patterned substrate 128 having patches ofimmobilization material, can be fabricated by any of the methodsdescribed herein. A preferred method is to prepare a substrate with apattern of photoreactive polymer. Subsets of mixed microparticles havingself-encoding marker/probe pairs can be individually deposited onseparate patches of the patterned substrate 128. Microparticles from thefirst subset, FIG. 3 d, are disposed within a first patch 124 on thesubstrate thereby forming a subarray on a patch 124 wherein themicroparticles of the subset are randomly distributed. An example of arandomly distributed microparticle 122 is illustrated. Microparticlesfrom the second subset, FIG. 3 e, can be deposited on a separate secondpatch 130. The patches are typically separated by a border 126 ofuncoated substrate. The width of this border 126 is preferably at leastas great as the diameter of an individual microparticle 122, morepreferably at least twice the diameter of the microparticle.

The process of depositing subsets of microparticles having uniqueself-encoding marker/probe pairs can be repeated or performedsimultaneously to provide the substrate with a desired number ofsubarrays. The location of a deposited subset or a pattern is typicallynoted to provide the user with information regarding the probe identityduring the detection step.

In some embodiments subsets of microparticles having self-encodingmarker/probe pairs can be deposited more than once on a substratethereby creating an array with multiple subarrays.

In another embodiment as illustrated in FIG. 4, immobilization materialcan be patterned on the substrate 130 to provide a plurality of patchesthat are sized to accommodate a desired number of microparticles, forexample, one microparticle per patch. Upon the disposing of a set ofmicroparticles having self-encoded marker/probe pairs individualmicroparticles can be spatially separated on the substrate. Preferably,the majority of the microparticles on the substrate are individuallyseparated wherein single microparticle patches 132 are displayed.However, it is possible that microparticle-devoid patches 134, doublemicroparticle patches 136, or patches containing more than twomicroparticles are displayed.

FIG. 5 shows a photomicrograph of a substrate having microparticlesseparated on the surface of a patterned substrate. The substrate waspatterned to create patches that were sized to accommodate approximatelyone microparticle per patch. FIG. 6 shows a photomicrograph of asubstrate having microparticles separated on the surface of a patternedsubstrate. The substrate was patterned to create patches that were sizedto accommodate multiple microparticles per patch.

In another embodiment, as shown in FIG. 7, immobilization material canbe patterned on the substrate to provide a plurality of patches whichare grouped at predetermined locations on the substrate. For example, asshown in FIG. 7 a, one group of patches is located in coordinate(X_(a),Y_(a)) on the substrate. Typically, these groups are separated bya border of uncoated substrate where microparticles will not beimmobilized preferably wider than the width of at least two patches. Asshown in FIG. 7 b a set or subset of microparticles having self-encodingmarker/probe pairs is deposited on the group of patches. In this examplethe patches are sized to accommodate approximately one microparticle perpatch and the number of patches within the group is greater than orequal to the number of unique microparticles having self-encodingmarker/probe pairs of the set or subset, therefore allowing the entireset or subset to be represented in one group of patches.

For example, the pattern can be printed on the surface using, forexample, a printing pin or a jet printer. In a preferred method, thepattern is generated by using a mask having a negative image of thepattern that is to be generated on the surface of the substrate. Use ofa mask for generating a pattern is particularly useful for fabricationof arrays wherein the matrix-forming polymer contains photoactivatablegroups and the activated groups are able to form bonds with thesubstrate and couple the polymer to the surface of the substrate

In yet another embodiment, a plurality of microparticles are coupled tothe substrate prior to application of microparticles intended to bearprobe molecules. According to this embodiment, a plurality of “unloaded”microparticles are deposited onto and coupled to the substrate prior toapplication of microparticles that are to be coupled to one or moreprobe molecules. This application of unloaded microparticles provides anintermediate layer between the substrate and microparticles that willbear probe molecules. According to this embodiment, this intermediatelayer can provide increased surface area for attachment of themarker/probe pairs, thereby increasing the loading density of the array.The coating of unloaded microparticles can cover some or the entiresubstrate surface. Additionally, “unloaded” microparticles can be usedto provide reasonable spacing to distinguish signals from theself-encoding marker and target marker on separate microparticles.

In some embodiments, it can be desirable to alter the spacing ofmicroparticles having self-encoding marker/probe pairs immobilized onthe substrate. This can be accomplished in any desirable manner, forexample, by providing microparticles that do not contain probemolecules. Preferably, the amount of space between the microparticlescan be adjusted by altering the ratio of the amount of polymer to theamount of microparticles to the amount of solvent during deposition ofthe microparticles. An increase in the space between the microparticlescan be achieved by increasing the amount polymer or decreasing theamount of microparticles, or a combination of the two.

In other embodiments, spacing of microparticles containing probemolecules can be altered by providing “unloaded” microparticles; thatis, microparticles that are not coupled to any probe molecules. Theseunloaded microparticles can be provided in a suitable ratio to thenumber of microparticles that are coupled to probe molecules, to achievethe desired spacing. According to the current invention, the ratio ofmicroparticles containing probe molecule to unloaded microparticles canbe in the range of 1:1 to 1:20.

In some embodiments of the invention, it can be desirable to providemultiple copies of a specific microparticle having a uniqueself-encoding marker/probe pair on a single array. This “redundancy” ofthe microparticle having a unique self-encoding marker/probe pair on thearray can provide increased sensitivity of the resulting array, since aprobe will be located at more than one location on the array. As aresult of this redundancy, a particular target will be capable ofbinding to at least one portion of the array. For example, in oneembodiment, the array can contain a particular microparticle having aunique self-encoding marker/probe pair that is immobilized on thesubstrate at 1 to 10 different locations. In this embodiment, detectionof the target associated with the redundant microparticles having aunique self-encoding marker/probe pair will take place at a number ofdifferent locations on the array.

In one embodiment, the sample suspected to contain a target is treatedto label the target. As used herein, “target marker”, refers to thedetectable moiety that is coupled or bound to the target and used todetermine the presence of the target, which can be present in a sample.As contemplated in this invention, a target marker comprises a moietythat is detectable using standard techniques known in the art. Examplesof suitable labels include, but are not limited to, fluorophores,phosphors, and radioisotopes.

When the target to be detected comprises RNA, the RNA target can belabeled using molecular biology techniques, such as in vitro run-offtranscription to generate a labeled RNA sample using RNA polymerases,for example T7, T3, or SP6 RNA polymerases. Kits for labeling RNA areavailable from various sources, for example, Ambion, Inc. (Austin,Tex.). This technique can be particularly useful in generating labeledRNA from, for example, a cDNA library that has been cloned into a vectorwith the appropriate promoters for RNA polymerase transcription.Techniques can also be used to generate labeled DNA, for example, nicktranslation, PCR amplification, random priming, or primer extension.These techniques can be useful for generating labeled DNA from forexample, cDNA libraries or genomic DNA libraries. Modified DNAnucleotides for use as labels can also be created from ReverseTranscriptase reactions. For example, an RNA sample, such as a polyA-RNAsample can be used as a template in a reaction containing ReverseTranscriptase, polyT oligonucleotide primer, and modified nucleotide togenerate labeled-cDNA. Techniques for labeling DNA can be found invarious technical references, for example, Current Protocols inMolecular Biology (Ausubel et al., ed., 1990, Greene Pub. Associates andWiley-Interscience: John Wiley, New York).

RNA and DNA targets can be labeled using modified nucleotides, forexample fluorophore-coupled nucleotides, such asFluorescein-5[6]-carboxyamidocaproyl-[5-(3-aminoallyl)uridine5′triphosphate (Sigma, St. Louis, Mo.), biotin-coupled nucleotides, suchas(N⁶-[N-(Biotyinyl-ε-aminocaproyl)-6-aminohexylcarbamoylmethyl]adenosine5′-triphosphate) or other modified nucleotides, for example,5-(3-aminoallyl)uridine 5′-triphosphate (Sigma, St. Louis, Mo.) in orderto enable detection of the DNA or RNA. Secondary fluorophore-coupledreagents, for example, Streptavidin-Cy3 (Caltag, Burlingame, Calif.) canbe used to for indirect detection of the modified nucleic acid.Radioactive nucleotides, for example ³²P-, ³³P-, and ³⁵S-labeledribonucleotides and deoxyribonucleotides can be incorporated into thetarget DNA or RNA present in a sample. These modified nucleotides canalso be used to label sample nucleic acids in other ways, for example,by 5′ or 3′ end-labeling with enzymes such as polynucleotide kinase orterminal transferase. Optionally, kits and instructions for couplingmodified nucleotides to nucleic acid samples can be obtainedcommercially from, for example, CALBIOCHEM (San Diego, Calif.). Labeledtarget can optionally be purified by methods such as gel filtration orpurification, spin columns, or selective precipitation.

In another embodiment, the sample is a protein sample suspected tocontain a protein target. The protein sample can be obtained from atissue sample, such as a biopsy, or from a fluid sample containingcells, for example blood or bone marrow, or from other body fluids, forexample, plasma, sweat, saliva, or urine. A protein sample can berecovered from body fluid by a variety of techniques, for example byprecipitation, filtration, or dialysis. A protein sample from cells, fortissue or body fluid, can be prepared by the lysis or solubilization ofcells in detergents, optionally using methods such as sonication orhomogenization. Ionic or non-ionic detergents can be used, for example,sodium dodecyl sulphate (SDS), Triton X-100, sodium deoxycholate orCHAPS. Cells can also be disrupted in the presence of chaotropicreagents, such as urea or guanidine salts. Other reagents can be addedto the detergent or chaotropic reagent, such as a buffer, for exampleTris or HEPES, and salts, for example KCl or NaCl. Other compounds canbe utilized which stabilize the protein sample, for example, proteaseinhibitors, such as PMSF, pepstatin, or EDTA. However, a variety ofmethods and buffer compositions are available for the lysis ordisruption of cells for protein extraction and are commonly known in theart. This information can be found in various references, for example,Current Protocols in Protein Science (Coligan et al., eds., 1996, JohnWiley & Sons, New York, N.Y.).

The protein sample is preferably labeled in such a way to enabledetection of the protein target and to retain the ability of the proteintarget to interact with the probe coupled to the microparticle. Reagentsare available that allow the coupling of a fluorophore to an amino acidresidue on a protein target. Amine-reactive groups, for example,succinimidyl esters, including sulfosuccinimidyl esters, isothiocyanatesand sulfonyl chlorides, or dichlorotriazines, aryl halides and acylazides are available as fluorophore probes and can be used for proteintarget labeling. A variety of these amine-reactive fluorophore probesare commercially available, for example, Alexa Fluor® 350 carboxylicacid, succinimidyl ester; 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester(BODIPY® 558/568, SE); and6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, succinimidyl ester(6-JOE, SE) (Molecular Probes, Eugene, Oreg.). In other circumstances itcan be desirable to label the protein target with a fluorescentthiol-reactive derivative, for example, N,N′-didansyl-L-cystine(Molecular Probes, Eugene, Oreg.). Alternatively other reagents, forexample fluorescent dyes containing a hydrazine group, an aromaticdiazonium salt, or an amine group can be used to label the proteinsample.

The protein target can also be coupled to a fluorescent protein, forexample the Green Fluorescent Protein (GFP), using commerciallyavailable bifunctional crosslinking reagents, for example NHS-ASA(Pierce Chemical, Rockford, Ill.). Other bifunctional crosslinkingagents that are reactive toward amine, sulfhydryl, carbohydrate,carboxyl and hydroxyl groups are commercially available (for example,Pierce Chemical, Rockford, Ill.) and can be used for coupling a proteinof interest to the protein target. The protein target can also becoupled to a primary reagent for secondary fluorophore detection. Insome embodiments, the protein can be modified by, for example,sulfo-NHS-biotin, and then coupled to a secondary fluorophore reagent,for example Streptavidin-Cy3.

Other methods of labeling a protein sample for detection are available.Such methods include, for example, protein iodination using ¹²⁵I andprotein phosphorylation using ³²P or ³³P and a protein kinase can beused.

The target can also be a particle, for example, a viral particle, aportion of a cell, a cell, or a subpopulation of cells. These targetscan display molecules on their surfaces that allow them to bind aparticular probe that can be present in the array. The binding of theprobe and the molecule present on a viral particle, a portion of a cell,a cell, or a subpopulation of cells can be useful in identifying andquantifying the target which can be present in a sample. For example,the array can be useful in determining the presence and quantity ofdifferent sub types of lymphocytes present in a sample. The viralparticle, cell, or a portion of a cell can be labeled for detection by avariety of means, for example, by labeling with an antibody that iscoupled to a fluorophore. The labeled antibody can be chosen to reactspecifically with a target, which can be a viral particle, a portion ofa cell, a cell, or a subpopulation of cells. Alternatively, the targetcan be labeled by incorporation of a fluorescent dye into a portion ofthe target, for example, the membrane. Such dyes, for example, PKH-67 GL(Sigma, St. Louis, Mo.) or a fluorescent lipophilic probe, CM-DiI(Molecular Probes, Eugene, Oreg.), are commercially available and can beused to label cells or other targets.

It is understood by one of skill in the art that there are a widevariety of techniques available for protein labeling and that thetechnique chosen can depend on the protein or proteins available in thesample targeted for labeling.

Given the description herein, one of skill in the art can select thedesired labeling scheme depending upon the target to be detected. Whilenucleic acids and proteins have been described with particularity, itwill be clear that the teaching herein can be applied to label samplessuspected to contain other types of targets, including, but not limitedto, small molecules and the like.

Once formed, the array of the invention can be used to detect targetsuspected to be contained in a sample. In use, a sample is modified toprovide labeled target, as described herein. The modified sample is thenapplied to the array, and the array and sample are maintained underconditions suitable to allow specific binding, for example, thehybridization of complementary nucleotides, between the probe andtarget, if present. Such specific binding conditions can be determinedusing techniques known in the art, depending upon the target to bedetected.

After binding, excess sample can be removed, for example, by washing,and the remaining hybridized targets can be interrogated. The arrays ofthe invention can be used to detect any desired target suspected to bepresent in a sample. Interrogation of a sample can involve one or morevisualization steps depending on the types of markers used for thetarget detection and microparticle detection. The label and detectionmarker can be chosen to allow detection of each using the sameinstrumentation, for example, a CCD camera or suitable fluorescencereader, although this is not required.

Typically, visualization of the results of an assay using the array ofthe invention involves one or more steps. Visualization of an analyticalsignal from the target marker, and visualization of the microparticleidentity via the self-encoding marker to determine the identity of probemolecule associated with the microparticle can be accomplished in onestep or more than one step. Visualization of the markers is dependent onthe type of markers used.

In one embodiment, both the target marker, associated with target, andself-encoding marker, associated with a particular microparticle, arevisualized using fluorescence microscopy. In this embodiment, presenceof the target marker can be determined using a first wavelengthassociated with the fluorescent particle chosen for the target marker.The location and identity of a particular microparticle can bedetermined by switching to a second wavelength that is associated withthe fluorescent particle chosen for the self-encoding marker coupled toor incorporated into the microparticle. Preferably, only thosemicroparticles giving rise to an analytical signal need to be detectedand decoded according to the invention.

The commercial fluorescent scanners described above have the capabilityto scan four different wavelengths, potentially allowing one to detectat least two self-encoding wavelengths and one target fluorescencewavelength. Commercially available self-encoded microparticles, such asthose manufactured by Luminex, are designed to work with 532 nm targetexcitation, one of the common wavelengths of commercial scanners. Onecommercial scanner system, the Packard BioSciences ScanArray 5000(Billerica, Mass.) has four wavelengths available for excitation (488nm, 543 nm, 594nm, and 633 nm). To create a simple self-encodingmicroparticle system it would be necessary to use labeled target at onewavelength, preferably 488 nm because many common oligonucleotide dyesexist for this wavelength (Fluorescein and its mimics) and because itwould be less likely to overlap with the self-encoding dyes. At leasttwo other wavelengths would then be used for the self-encoding dyes. Oneexample would be to label the microparticles with a Texas Red dye(T-6134, Molecular Probes, Eugene, Oreg., excitation 596/emission 620nm) which has little or no overlap with fluorescein, and a longerwavelength dye such as BODIPY 630/650 (D-10000, Molecular Probes,Eugene, Oreg., excitation 630/emission 650 nm). Based on itsfluorescence absorbance and emission spectra, BODIPY 630/650 shouldadsorb a small fraction of the 596 nm light exciting the Texas Red dye,but none of the 488 nm target wavelength. To determine the correspondingspectral profile, the fluorescence at 594 and 633 nm excitation of eachbatch of self-encoded microparticles with a given ratio of TexasRed/BODIPY dyes would be ascertained prior to the assembly of themicroarray and the overlap accounted for. This system could potentiallybe extended to include dyes at the 543 nm wavelength as well, becausethere is little light adsorbed by fluorescein derivatives at thatwavelength. Given at least two and potentially three different dyes atdifferent ratios, the number of uniquely self-encoded microparticles islarge, at least 1000 (10 different ratios of each dye).

Visualization techniques can include, for example, spectrometrictechniques such as UV/VIS, IR, fluorescence, chemiluminescence, massspectrometry, or other methods known in the art, or combinationsthereof.

It will be apparent to those skilled in the art that changes may be madeto embodiments as described herein without deviating from the scope ofthe claimed invention. The following examples are provided toillustrate, but are not intended to limit, the present invention.

EXAMPLES Example 1

Polypropylene and silanated glass slides (1×3 in.×1 mm) were used in thepreparation of substrates in array fabrication. Glass microscope slideswere obtained from Erie Scientific, Portsmith, N.H. (catalog # 2950-W).These soda lime glass microscope slides were silane treated by dippingin a mixture of 1% v/v p-tolyldimethylchlorosilane (T-Silane) and 1% v/vn-decyldimethylchlorosilane (D-Silane, United Chemical Technologies,Bristol, Pa.), each in acetone, for 1 minute. After air drying, theslides were cured in an oven at 120° C. for one hour. The slides werethen washed with acetone followed by dipping in deionized water. Theslides were further dried in an oven for 5-10 minutes. Polypropyleneslides were obtained from Cadillac Plastics (Minneapolis, Minn.).

The silanated glass or polypropylene slides were then washed in acetoneor isopropanol. The washed slides were then dip-coated in a 1 mg/mlsolution of photo-Polystyrene (pPS) in toluene. pPS was prepared bypulverizing 3.05 g Polystyrene MW 500 (Polysciences Inc., Warrington,Pa.) with mortar and pestle and then dissolving the pulverizedpolystyrene in approximately 50 mL of carbon disulfide (Aldrich,Milwaukee, Wis.). A reaction flask was flushed with argon gas to ensurean anhydrous atmosphere and the dissolved polystyrene was placed in theflask. 2.91 g aluminum chloride (Aldrich, Milwaukee, Wis.) was added tothe dissolved polystyrene, followed by the dropwise addition of then 2.5mL of benzoyl chloride (Aldrich, Milwaukee, Wis.). The reaction mixturewas then stirred at room temperature under Argon for 30 minutes,followed by refluxing the mixture for twelve hours. The mixture was thencooled and the solvent decanted off the reacted polymer. The polymer wastriturated in methanol until a yellow solid was formed. This solid waswashed with methanol and then dissolved in choloroform and precipitatedfrom solution by methanol addition. This process yielded 1.16 g ofyellow solid. Alternatively, the slides can be dip-coated inbenzoylpolystyrene (Cat. #7917 Lancaster Synthesis, Windham, N.H.). Thecoated slides were air dried and irradiated for two (2) minutes withbroad spectrum ultraviolet light (320-390 nm) using a Dymax LightWelderPC-2 (Dymax Engineering Adhesives, Torrington, Conn.) having a typicalpower output of 2 mW/cm². The lamp was positioned approximately 10 cmfrom the slides. Following irradiation, the slides were rinsed withtoluene to remove unbound pPS. The pPS-coated slides were then immersedin an aqueous solution of 5 mg/mL biotinylated triblock (Dow PolyglycolB40-2500) for five (5) minutes, followed by irradiation for two (2)minutes and rinsing in isopropanol. The pPS-coated surface bound thebiotinylated triblock surfactant effectively, creating a surface withuniformly distributed biotin moieties as determined bystreptavidin-coated microparticle binding. Uniform distribution ofbiotin moieties was also determined by binding of fluorescently labeledavidin solution and determining the fluorescence pattern on the slides.

Magnetic, polystyrene-encapsulated microparticles, coupled to aflurorescent blue dye (excitation/emission maxima of 490/515 nm) andstreptavidin were obtained from Bangs Laboratories (Product # CM01F;Fishers, Ind.). The streptavidin concentration was measured bybiotin-conjugate binding and was determined to be 9.86 μgbiotin-alkaline phosphatase/mg microbeads and 0.77 μg biotin-FITC/mgmicrobeads per manufacturer's measurement. The microparticles had adiameter of 0.96 μm and a density of 1.7 g/cm³ (1.305e+12microparticles/g). The prepared microparticles were washed once inphosphate buffered saline, pH 4.6 (PBS) including 0.1% bovine serumalbumin (BSA) and 0.1% Tween 20 (PBS-B-T). After washing, themicroparticles were resuspended in PBS-B-T. The biotinylated slide,prepared as described above, was soaked with PBS-B-T for thirty (30)minutes at room temperature. The phosphate buffer was then gently rinsedaway with deionized water and centrifuged dry.

The washed microparticles were then added to the biotinylated slide at aconcentration of 1 mg/mL (approximately 1.305 e+9 microparticles/ml) Themicroparticles and slide were incubated at room temperature for 20 to 30minutes with gentle mixing by hand, with a three-dimentional motion,every few minutes.

Example 2

An array was formed by immobilizing microparticles on the substrate inthe presence of a crosslinking agent, where the microparticles affinitybound the substrate and were coupled together by the presence of acrosslinking agent. pPS-coated slides, as prepared in Example 1, andstreptavidin- and fluorophore-coated microparticles, as described inExample 1, were used in this example.

Amine-reactive N-hydroxysuccinimide (NHS) ester cross-linking reagent,disuccinimidyl suberate (DSS; Cat.#21555; Pierce Chemical, Rockford,Ill.) stock solution was prepared by dissolving DSS in dimethylformamide(DMF) at a concentration of 10 mg/mL. DSS stock solution was thendiluted in PBS to a concentrations of 0.05, 0.025, 0.25, and 0.38 mg/mLDSS. The PBS/DSS solution was then added to the washed microparticlesuspension (as described in Example 1) and the microparticles had aconcentration of 1 mg/ml in the final solution. The final concentrationof DMF was kept below 5%, to avoid potential solvent damage topolystyrene microparticles and streptavidin.

The PBS/DSS/microparticle mixture was added as a slurry to thebiotinylated slide. The PBS/DSS/microparticles mixture and biotinylatedslide were incubated at room temperature for 20 to 30 minutes withgentle mixing by hand, with a three-dimentional motion, every fewminutes. This step allowed the DSS to crosslink covalently attachedstreptavidin molecules on different microparticles, thereby formingmicrophere-microparticle linkages. DSS also crosslinked streptavidinmolecules on the same microparticle using available primary amines ofthe streptavidin molecules. Streptavidin molecules on the microparticlesalso affinity bound the biotin on the slide surface and the DSScrosslinking did not reduce this affinity binding.

After incubation of the microparticles and slide, the slide is gentlywashed with a 0.1% Tween 20 solution, followed by rinsing with deionizedwater. The slide is then centrifuged dry in a slide rack holder in aclinical centrifuge (rotor radius=8 cm) at a speed in the range of500-1000 RPM. The prepared substrate was stored until use under ambientconditions.

Example 3

An array was formed by immobilizing microparticles on the substrate byfirst providing a monolayer of microparticles on the substrate and thenproviding a subsequent layer of microparticles that became crosslinkedin the presence of a crosslinking agent. pPS-coated slides, as preparedin Example 1, and streptavidin- and fluorophore-coated microparticles,as described in Example 1, were used in this example. The DSScrosslinking agent, as prepared in Example 2, was used in this example.

Microparticles coated with streptavidin were applied to the substrateand incubated to allow the streptavidin to non-covalently affinity bindto the biotin provided on the substrate. This coating provides amonolayer of streptavidin-coated microparticles. The coated substratewas washed to remove unbound microparticles. Thereafter, a solutioncontaining microparticles was applied as a slurry to the substratecontaining a monolayer of streptavidin coated microparticles.

The substrate and slurry were incubated at room temperature for 20 to 30minutes with gentle mixing by hand, with a three-dimentional motion,every few minutes. After incubation of the microparticles and slide, theslide is gently washed with a 0.1% Tween 20 solution, followed byrinsing with deionized water. The slide is then centrifuged dry in aslide rack holder in a clinical centrifuge (rotor radius =8 cm) at aspeed in the range of 500-1000 RPM. The prepared substrate was storeduntil use under ambient conditions room temperature.

Example 4

As shown in this example, an array was fabricated without pretreatmentof the substrate surface. Microparticles were prepared by coupling aprobe to the microparticle, followed by preparation of a slurrycontaining a photoreactive polymer and the probe-coupled microparticles.These prepared microparticles were then applied to a substrate to forman array.

Magnetic, streptavidin-coated fluorescent microparticles (0.96 μm) wereobtained from Bangs Labs, (Fishers, Ind.) as described in Examples 1-3.The microparticles, were washed twice with deionized water andresuspended in 25 mM phosphate buffered saline, pH 8, at 10 mg/mL.

The streptavidin-coated microparticles were coupled to oligonucleotideBN30. BN30 is a 30-mer with a 5′ biotin modification and a 3′ amine(Integrated DNA Technologies, Coralville, Iowa). Coupling was performedusing 5 nmole/ml of BN30 and 1 mg/ml of microparticles (five-fold excessof biotinylated oligonucleotide, based upon the supplier's stated biotinbinding capacity) in 25 mM PBS, pH 8. The coated microparticles andoligonucleotide were incubated for 30 minutes at room temperature withgentle agitation. After incubation, the microparticles were washed withdeionized water and resuspended at 20 mg/ml in deionized water.

A slurry of 37 mg/ml photoreactive poly(vinylpyrrolidone) (PV01;SurModics, Inc., Eden Prairie, Minn.) in water was combined with themicroparticle solution at a ratio of 9:1 (10× dilution of microparticlesolution in PV01). The slurry was printed onto an acrylic surface(obtained from Cadillac Plastics, Minneapolis, Minn.) using a MicrogridII arrayer (Biorobotics, Inc. Cambridge, UK) with an average spot sizeof 100 μm and a center to center spacing of 250 μm, providingapproximately 16 spots/mm². The printed substrate was irradiated for two(2) minutes as described in Example 1 with the addition of a beneath acut-off filter (315 nm) to avoid potential nucleic acid damage, whilegelling the PV01 solution. After irradiation, the substrate was rinsedwith 1× PBS, 0.1% Tween 20.

The arrays prepared by the above method were stable to washing andtouching, while samples that were not irradiated were not stable.Fluorescence scanning and light microscopy were used to determine thepresence and location of the microparticles

Sample containing nucleic acid complementary to probes of the arrayprepared as described above were interrogated as follows. Samplecontaining target nucleic acid was applied to the array and incubated.As described in Example 5.

Target was detected on the array with a ScanArray 5000 fluorescencescanner (Packard Bioscience, Billerica, Mass.).

Example 5

An array was fabricated by applying a suspension of microparticles to asubstrate having a surface patterned with a polymer. Hydrophilicmicroparticles, which can be coupled to oligonucleotides or otherbiomolecules, were captured in the polymer when in aqueous solution andremain trapped there upon drying. A polymer pattern was formedapproximately of the same size order as the bead diameter.Microparticles in the size range of 100 nm to 50 μm can be used forformation of an array, however, a preferred range is about 1 to 50 μm.Separate polymer spots were formed and there was approximately onemicroparticle per polymer spot.

Using this method a DNA microarray can be created using self-encodedmicroparticles wherein every unique self-encoded microparticle iscoupled to a unique oligonucleotide sequence. Mixing variousself-encoded oligonucleotide-coupled microparticles in the suspensionwould result in a random array of single microparticles, eachmicroparticle/spot containing a different oligonucleotide sequence thatcan be probed.

An aqueous solution of 10 mg/ml photoreactive poly(vinylpyrrolidone)(PV01; SurModics, Inc., Eden Prairie, Minn.) was coated onto ahydrophobic glass slide as detailed in Example 1. Alternatively, thePV01 solution can be allowed to dry down in air on the slide. The coatedslide was then irradiated for two minutes with ultraviolet light asdetailed in Example 5 (Dymax Lightwelder) through a patterned mask(fabricated at the University of Minnesota, Minneapolis, Minn.), withstructural features on the order of 1-100 Tm. The mask used has fourarrays of different sized patterns (200 μm spots, 500 μm center tocenter spacing, 100 μm spots, 250 μm center to center spacing, 50 μmspots, 100 μm center to center spacing, and 10 μm spots, 50 μm center tocenter spacing) and any pattern can be used for preparation of thearray. Each of the four arrays measures 7.4×7.4 mm².

Irradiation served both to crosslink the PV01 to itself and tocovalently bond it to the surface of the slide. After irradiation, theslide was washed with deionized water and isopropanol to remove excessPV01 from the non-irradiated areas of the pattern.

A suspension of 1.0 mg/ml streptavidin-coated silica microparticles (Catno. SS06N, Bangs Laboratories, Fishers, Ind.) was placed over thepatterned area of the slide created above in a 0.5M NaCl aqueoussolution. The microparticles were allowed to settle for 15 minutes,after which a brief washing step with water was performed to removemicroparticles which had not entered the polymeric matrix. At this pointthe microparticles were imaged by visible microscopy as shown in FIG. 5to ensure that single microparticles formed in the array.

When utilizing oligonucleotide-coupled microparticles immobilized in thepolymer pattern, the array can be washed with deionized water and then1× PBS with 0.5% Tween-20. The array can then be incubated at 45° C. for2 hours in 4×SSC and then subjected to a final deionized water wash. Thethoroughly washed arrays can then be subjected to a standard protocolfor hybridization of a target nucleic acid.

A typical hybridization can include, for example, 2.5 TL of a 33 fMsolution of a fluorophore-coupled target nucleic acid in hybridizationbuffer (5×SSC, 0.1% SDS, 0.1 mg/ml salmon sperm DNA) per cm² (arrayarea) placed between a coverslip and the array surface. The slides canthen be placed in hybridization chambers and heated in a water bath at45° C. for 2 hours. The coverslips can then be removed with a stream of4×SSC buffer and the slides can then be washed with 2×SSC/0.1% SDS forfive minutes at 45° C., followed by a 0.2×SSC wash for one minute atroom temperature, and finally a wash of 0.1×SSC for one minute at roomtemperature. The slides could then be spun dry and the targetoligonucleotide detected by methods described below.

Fluorescent target oligonucleotides can be detected on the microarraywith either a commercial fluorescence microscope (Olympus BX 60, Tokyo,Japan) or a commercial fluorescence scanner using confocal fluorescencemicroscopy (Packard BioSciences, ScanArray 5000, Billerica, Mass.). Themicroparticles used in fabrication of the arrays can be chosen accordingto the resolution capabilities of the detection equipment. Currently, astandard fluorescence microscope can detect a microparticle in the sizerange of 1-50 Tm diameter. Fluorescence scanners currently possess notgreater than 5 Tm resolution. A 5 Tm area is illuminated in the focalplane of the slide and represents one pixel. Single microparticles wouldbe detectable if the diameter is approximately 10 Tm.

Example 6

Different matrix-forming materials were used to immobilizemicroparticles on a substrate illustrating the use of various reactivepolymers for the fabrication of arrays. First, different polymers weredisposed and treated on a substrate to provide immobilization materialon the surface of the substrate. This was followed by disposing themicroparticles on the substrates which were immobilized via the treatedpolymers. Four different photoreactive polymers were used to immobilize9.9 μm diameter silica microparticles on substrates. These photoreactivepolymers PA04, PA05, PV01, and PV05 are all commercially available fromSurModics, Inc. (Eden Prairie, Minn.). PA04 and PA05 are copolymers ofacrylamide (AA) and N-[3-(4-Benzoylbenzamido)propyl]methacrylamide(BBA-APMA) with differing ratios of BBA-APMA:AA. Similarly PV01 and PV05are copolymers of vinylpyrrolidone (VP) andN-[3-(4-Benzoylbenzamido)propyl]methacrylamide (BBA-APMA) with differingrations of BBA-APMA:VP. Additionally a control material, PR03(ethylene(4-benzoylbenzyldimethylammonium)dibromide; SurModics, Inc.Eden Prairie, Minn.; described in U.S. Pat. No. 5,714,360 to Swan etal., issued 3 Feb., 1998, commonly owned by the assignee of the presentinvention, the disclosure of which is incorporated herein in itsentirety), a non-polymeric photoreactive compound, was evaluated.

Each photoreactive compound was dissolved in deionized water at aconcentration of 2.5 mg/ml. Each solution was printed with 25 gaugedisposable needles (PrecisionGlide Needles, Becton Dickinson and Co.,Franklin Lakes, N.J.) and an x-y programmable stage (CAMM-3, RolandDigital Group, Irvine, Calif.) onto glass microscope slides which hadbeen functionalized with silanes, as in Example 1, and onto acrylicslides (Cadillac Plastics, Minneapolis, Minn.). This printing forms apattern of approximately 300-400 μm diameter spots on the substrates.

The patterned slides were irradiated for two minutes with ultravioletlight as detailed in Example 1. After irradiation, 500 μl of an aqueoussolution of 2 mg/ml 9.9 μm diameter silica microparticles (SS06N, BangsLaboratories, Fisher, Ind.) was placed over the patterned area for oneminute to allow the microparticles to become immobilized by the polymermatrices. The slides were then rinsed with 0.1% v/v Tween-20 aqueoussolution to remove any free microparticles.

Following this, the substrate was washed to remove loosely boundmicrospheres. The substrate was washed three times with 1× PBS (pH 7.4)with 0.1% v/v Tween-20, and then rinsed with deionized water. At thispoint, each was imaged with a fluorescence microscope (Olympus BX 60,Tokyo, Japan), to determine microsphere loss in the variousphotoreactive polymer matrices. After imaging, the substrates wereincubated in a solution of DB02 wash buffer (Surmodics Inc. EdenPrairie, Minn.) for 1 hour at 50° C., followed by two rinses withdeionized water. The substrates were then incubated in a solutionof4×SSC/0.1% SDS for two hours at 50° C. and rinsed in deionized water.This was the final step at which imaging was done to evaluate therespective polymers. Results are summarized in Table 3. TABLE 3 Presenceof Presence of Presence of microparticles microparticles Photoreactivemicroparticles after PBS-Tween after high salt Compound Substrate beforewashes wash wash (4× SSC) PA04 Glass Present Some loss Significant lossPA04 Acrylic Present Some loss No loss PA05 Glass Present Some loss Noloss PA05 Acrylic Present No loss Some loss * PV01 Glass Present No lossNo loss PV01 Acrylic Present No loss No loss PV05 Glass Present No lossNo loss PV05 Acrylic Present No loss No loss PR03 - non- Glass PresentSome loss Complete loss polymer control PR03 - non- Acrylic Present Someloss Complete loss polymer control* Sample was touching another slide during the wash.

1. An array comprising: a) a substrate; b) a plurality of microparticlesrandomly immobilized on the wherein each microparticle comprises aself-encoding marker and a probe couple to the microparticle, whereineach self-encoding marker and probe comprises a unique self-encodingmarker/probe pair, and wherein the probe is configured and arranged tospecifically bind a target present in a sample; and c) an immobilizationmaterial which immobilizes the microparticles on the substrate; whereinthe immobilization material comprises a reactive polymer, the reactivepolymer bound to the substrate, and the microparticles immobilized onthe substrate via the reactive polymer; wherein the reactive polymercomprises a photoreactive polymer having at least one photoreactivegroup.
 2. The array of claim 1 wherein the photoreactive group isselected consisting of aryl ketones, arylazides, acyl azides, sulfonylazides, phosphoryl azides, diazoalkanes, diazoketones, diazoacetates,and ketenes.
 3. The array of claim 1 the reactive polymer is selectedfrom the group consisting of functionalized polyacrylamide,polymethacrylamide, polyvinylpyrrolidone, polyacrylic acid, polyethyleneglycol, polyvinyl alcohol, poly(HEMA), copolymers thereof, andcombinations thereof.
 4. The array of claim 1 wherein the reactivepolymer is selected from the group consisting of photoreactivecopolymers consisting of vinylpyrolidone andN-[3-(4-Benzoylbenzamido)propyl]methacrylamide (BBA-APMA); andacrylamide and BBA-APMA.
 5. The array of claim 1 wherein the reactivepolymer comprises polymers selected from the group consisting offunctionalized polysaccharides, glycosaminoglycans, polypeptides, andcombinations thereof.
 6. The array of claim 1 wherein the immobilizationmaterial is patterned on the substrate thereby forming patches ofimmobilization material on the substrate.
 7. The array of claim 6wherein the microparticles are immobilized on the substrate via thepatches of immobilization material.
 8. The array of claim 7 wherein theplurality of microparticles comprises more than one subset ofmicroparticles having unique self-encoding marker/probe pairs and thesubsets of microparticles are individually disposed on separate patchesof immobilization material on the substrate.
 9. The array of claim 1wherein the microparticle further comprises a coating of a reactivecompound, wherein the reactive compound is used to couple themicroparticles to the immobilization material, the self-encoding marker,the probe, or any combination of the above.
 10. The array of claim 9wherein the reactive compound includes reactive groups selected from thegroup consisting of carboxylic acids, sulfonic acids, phosphoric acids,phosphonic acids, aldehydes groups, amine groups, thiol groups,thiol-reactive groups, and epoxide groups.
 11. The array of claim 1wherein the probe is a nucleic acid probe.
 12. The array of claim 1wherein the probe is an antibody probe.
 13. The array of claim 1 whereinthe self-encoding probe comprises at least one detectable particle andwherein the at least one detectable particle is coupled to themicroparticle.
 14. The array of claim 13 wherein the detectable particleis selected from the group consisting of fluorphores, quantum dots,radioisotopes, and magnetic particles.
 15. The array of claim 14 whereinthe self-encoding marker comprises a combination of detectableparticles.